Experiment 1: Microscope

type of microscope used in lab

brightfield (projects bright light)

binocular (ocular for each eye with a mag lens)

compound (objectives lenses to get mag up to 1000x)

Magnification

objective x ocular

objective: magnifies specimen, real image

ocular: magnifies real image to make virtual image which we see

total magnification

obj (highest is 100x) x ocular (10x)

1000x

what is magnification limited by (a property)

resolution

what is resolving power

closest spacing btwn 2 objects in a microscope where u can still see 2 things as separate

what does resolving power depend on

wavelength of light and property of obj lens (numerical aperture)

max resolution for any light microscope

0.2 microns (so u can still see. them as separate entities)

bact cells are abt 1 micron so we can see them as separate entitites

refracture of light

light enters condensor —> light scattered —> doesnt reach lens —> bad resolution

so less light in lens = bad resolution

why do we use immersion oil for 100x

oil has same refractive index as glass slide and obj lens

oil reduces amt of light thats scattered so better resolution

what are the general shapes of bacteria

1. coccus (main)

2. bacillus (main)

other

3. spirillium

4. caulobacter (stalk)

5. corynebacterium (club-shaped)

6. vibrio (comma shaped)

Cell shape: Coccus

Arrangements?

1. coccus (one)

2. diplococci (two)

3. streptococci (chains)

4. staphylococci (cluster)

5. tetrad (square)

Cell shape: Bacillus

Arrangements?

1. bacillus (one)

2. diplobacillus (two)

3. streptobacillus (chains)

4. coccobacillus (round rod

Cell shape: other

Arrangements?

1. filamentous (long thread like)

2. spirillum (spiral or helical)

3. vibrio (comma)

how to leave microscope when leaving the lab (3)

1. 4x lens

2. power cord wrapped around back

3. plastic cover on

Exp 1 learning objectives

1. demonstrate how to use microscope (know how to care for it, parts, f(x)

2. perform microscopy and observe from 10-100x

3. examine cells and describe how they look

Exp 1 Materials (individual)

1. compound microscope (on bench)

2. prepared slides (one cocci one bacilli)

3. immersion oil

4. kim wipes

5. lens cleaner

What should you do if the focused slide image does not move when moving the slide's position before oil immersion?

Go back to 10X and refocus

When should the coarse focus knob be used?

When using the 10X objective lens for large adjustments. 

What happens if two bacterial cells are closer than 0.2 microns when observed under a light microscope?

They appear as a single entity.

When using the 100X oil immersion objective, what minimal adjustment should be made?

Fine-tune with the fine focus knob. 

How can the total magnification of a microscope be calculated?

What should you do if oil accidentally contacts the 40X objective?

Clean the lens immediately with lens cleaner and a kimwipe

What is the main advantage of using a binocular microscope?

  It enables viewing with both eyes as there is an ocular for each eye with a magnifying lens for better image 

When using the 100x objective lens, which additional component is required for optimal results?

 Immersion oil 

What is the main reason for starting with low objectives (e.g., 10X) when examining a slide?

To easily scan the slide and locate the area of interest. 

What is the correct sequence for focusing a slide from low to high magnification?

4x → 10x → 40x → 100x

What is the correct procedure for lifting and carrying the microscope?

Using the arm handle and finger grip

What could be the problem if no light is visible through the eyepiece, even when the microscope is plugged in?

The power/intensity switch is off. 

What should you do if you feel pressure or resistance when rotating the 100x oil immersion objective into position?

Call your demonstrator for advice 

What is the correct first step when preparing to use the microscope?

Remove the plastic cover and position the microscope

What is the function of the objective lens in a compound microscope?

 To magnify the specimen and produce a real image. 

Which part of the microscope should be adjusted to change the amount of light that reaches the specimen?

 Iris Diaphragm

Identify the parts of the microscope that directly interact with the microscope slide.

 Slide holder and Stage

how to focus on a smear

1. lower stage w coarse adjustment (atleast one full rotation)

2. put slide, smear side up

3. move L R B F w stage adj knobs

4. use nosepiece to rotate to 10x, rotate iris diagrpahm to 10x also, coarse to adjust and focus

5. FOCUS AWAY from specimen

6. then 40x, same rules

7. then move halfway and put oi on slide and slide to 100x (only fine adj with 100x)

Results: Cocci Demo

Staphylococci

Coccus (round)

1000x

Results: Bacillus Demo

Streptobacillus

Bacillus (rod)

1000x

What is the purpose of immersion oil in microscopy?

lowers the refraction = better resolution

When youre focused under 40x, why shouldnt u lower the stage to put oil on?

Will lose the focus and you have to go back to 10x

Instead just go midway between 40x and 100x and put oil